Over the last thirty years, polymerase chain reaction (PCR) has become a widely used biological assay in both laboratory and clinical settings. Subsequently, the advent of real-time PCR enabled the automation of both the amplification and detection steps. Various bench-top real-time PCR devices with high-throughput and multiplexing features have been developed. More recently, fully automated systems that cover sample preparation, DNA extraction, PCR amplification and detection have also been developed. These devices enable the rapid, accurate and specific diagnosis of a host of bacterial and viral pathogens.
However, there is an acute and unmet need for such point-of-care (POC) diagnosis in developing countries where centralized laboratories are lacking. Portability is a key POC feature for PCR devices and would impact the design of optical modules for such systems. A portable system would require the optical module to (i) have non-movable components, (ii) occupy a smaller footprint, (iii) be battery operated, (iv) have sufficient detection sensitivity, and (v) provide moderate throughput and multiplexing.
Bench-top PCR devices designed for centralized laboratories usually employ movable optical components so that the detector (e.g. charge-coupled devices (CCDs), photomultiplier tubes (PMTs), photodiodes) and excitation light source can be automatically positioned over different regions of a 96- or 384-well plate using an x-y motor. Bench-top PCR devices also employ motorized excitation, dichroic and emission filter wheels to automate the detection of multiple probes of specific spectral bandwidths.
However, the absence of an x-y motor would imply that the photodetector has a field of view as large as a 96- or 384-well plate. Also, the excitation light would need to be projected over a large area and, as such, the intensity of light per unit area must be sufficiently high to detect the fluorescent probes. This necessitates the use of high-powered tungsten or mercury lamps, which would detract from the goal of developing a portable POC PCR device. With the advent of high-power (greater than five watts), high brightness and compact light-emitting diode (LED) light sources in both ultraviolet (UV) and visible ranges of wavelengths, an efficient and battery-powered (approximately twelve volts) alternative to conventional light sources has been presented.
Further, different strategies have been implemented in developing a non-motorized optical detection system. For example, a single blue LED has been utilized to simultaneously excite fluorescence-conjugated probes with emission wavelengths of 530, 640 and 705 nm by fluorescence resonance energy transfer (FRET). The emission spectrum is then collected by three corresponding photodiodes via a cascade of emission bandpass filters and beam splitters. While this optical scheme is inexpensive, the collection efficiency of the emitted signal is limited to ˜40-50% due to transmission losses.
One conventional solution proposed the use of a spectrometer for detecting and resolving emission wavelengths of fluorescence-conjugated DNA probes. The benefit of using a spectrometer is that it offers excellent wavelength resolution that is of importance in a multiplexed PCR setting. However, it lacks spatial resolution, and this necessitates the use of a motorized carousel to sequentially position the DNA samples in direct view of the optical fiber connected to the spectrometer. Alternatively, a 1- or 2-dimensional scanner needs to be incorporated to achieve spatial resolution.
Another conventional solution developed a CCD-based fluorimeter for real-time PCR. A 470-nm LED was used as an excitation light source, but instead of cascading the emission filters, a rotating disk was implemented with six optical filters to provide six fluorescence detection channels at 530, 560, 610, 640, 670 and 710 nm. Although the issue of limited collection efficiency is addressed, this design is more complex given the need to motorize the rotating disk.
A further conventional device utilizes a compact and modularized cartridge system comprising a PCR reaction chamber, a thermal cycling module, four LED excitation lights and corresponding photodiodes for detection. However, each cartridge processes only one patient sample and has a high cost. In addition, the integrated cartridge limits the scope of multiplexing to just optical, rather than a combination of spatial and optical multiplexing.
An additional solution implemented a real-time PCR optical detection system incorporating a laser, a filter cube, a photo-detector and a 1×4 fiber optical switch so that the fluorescence signal can be acquired from four different reaction sites. Such a design appears well-suited for a portable PCR system, whereby there is a spatial multiplexing of four and the entire system does not have any movable parts. However, there is no optical multiplexing since the laser produces excitation at a specific wavelength, and both the dichroic mirror and emission filter have bandpass properties.
A further system employs a real-time PCR device that uses a nested PCR, whereby amplified DNA samples are delivered to a multi-well chip for a second round of real-time PCR amplification. Each well is preloaded with a specific primer pair and SYBR green intercalating dye for the detection of a single DNA target using a non-movable CCD-based fluorescence imaging system. Although this system offers high spatial multiplexing, it has only a single optical channel for detection.
Thus, what is needed is a portable system with an optical module to (i) have non-movable components, (ii) occupy a smaller footprint, (iii) be battery operated, (iv) have sufficient detection sensitivity, and (v) provide moderate throughput and multiplexing. Furthermore, other desirable features and characteristics will become apparent from the subsequent detailed description and the appended claims, taken in conjunction with the accompanying drawings and this background of the disclosure.